Quantification of the strength of cell-cell adhesion: the capture of tumor cells by activated murine macrophages proceeds through two distinct stages.

The binding of tumor cells by macrophages activated with Bacillus Calmette-Guerin is a necessary step toward destruction of those cells. Although several characteristics of the interaction have been defined, little is known of how the actual binding process develops. We used a technique to quantify the forces required to disrupt cell-cell interactions. Over a range of applied relative centrifugal forces, the majority of targets that bound to the activated macrophages fell on two distinct plateaus. Approximately 90% of added targets were bound to the monolayers of macrophages over the range of 1 to 100 X G; 25 to 30% remained bound from 1200 X G to 1500 X G. Two strengths of binding, termed weak and strong binding, respectively, were thus defined on the basis of these curves. Strong binding developed only between activated macrophages and tumor cells. By contrast, weak interactions occurred between either activated or nonactivated macrophages and neoplastic or non-neoplastic target cells. The strong binding required time (60 to 90 min), metabolic activity by the macrophages, and trypsin-sensitive surface structures on the macrophages for development, whereas the weak interaction occurred rapidly and required none of these. Additional evidence indicated the weak binding developed into strong when activated macrophages bound neoplastic cells. This stabilization increased the strength of force to separate tumor cells from the macrophages at least approximately 15 fold (i.e., from approximately 16 mu dynes/cell to approximately 240 mu dynes/cell). Of note, the development of strong binding of antibody-coated targets had distinct requirements for establishment. Taken together, the data suggest the stabilization of binding (i.e., the development of weak into strong binding) leading to effective cell-cell interaction is a complex and dynamic process that may vary depending upon the recognition system involved.

The binding of BCG-activated macrophages to tumor targets stimulates secretion of cytolytic factor.

The binding of neoplastic targets and the secretion of a potent cytolytic protease (CF) by BCG-activated macrophages have previously been shown to be independent functions, both of which are necessary for completion of macrophage-mediated cytolysis. The present studies demonstrate that secretion of CF is triggered by the binding of neoplastic targets to BCG-activated macrophages. The binding of tumor targets, but not of normal lymphocytes, resulted in enhanced secretion of CF from BCG-activated macrophages, although not from macrophages elicited by thioglycolate broth. Dead or metabolically inactive tumor targets, as well as membrane preparations of tumor targets, induced secretion of CF from BCG-activated macrophages. The blocking of macrophage-target binding with a porous filter prevented augmented secretion of CF. Appreciable secretion of CF occurred in as little as 30 min after addition of tumor targets to BCG macrophages. Binding did not induce a generalized increase in secretion of neutral proteases by BCG macrophages, since secretion of plasminogen activator was actually decreased after the binding of P815 targets. The data suggest the selective binding of BCG-activated murine macrophages to neoplastic targets triggers the secretion of a potent CF.

Binding of bacillus Calmette-Guérin-activated macrophages to tumor targets. Selective inhibition by membrane preparations from homologous and heterologous neoplastic cells.

The binding of tumor cells by activated macrophages is an initial and necessary event in the cytolysis of these targets. The data here indicate that membrane preparations from RL sigma 1 leukemia targets, EL-4 lymphoma targets, and P815 mastocytoma targets each inhibited binding of its homologous target to bacillus Calmette-Guérin (BCG)-activated murine macrophages in a dose-dependent fashion. Similar amounts of membrane from lymphocytes did not alter binding of the three neoplastic target to BCG-macrophages. Membranes of the three targets also inhibited binding of the heterologous neoplastic targets. Inhibitory activity of membrane preparations from P815, EL-4, and RL sigma 1 targets could be adsorbed by incubation of limiting concentrations of the membrane preparations with BCG-activated macrophages but not with thioglycollate broth-elicited macrophages. Exposure of BCG macrophages to membrane preparations from RL sigma 1, FL-4, or P815 targets inhibited subsequent cytolysis of the three targets. Inhibitory activity was increased in preparations enriched for plasma membrane. The data suggest that binding of three murine, nonadherent neoplastic targets to BCG-activated murine macrophages is mediated, in part, by recognition structures present within the plasma membranes of the three targets.

Interaction of cytotoxic T lymphocytes with target cells. I. Specific inhibition by detergent-solubilized, partially purified mouse histocompatibility antigens.

H-2 antigens from EL4 (H-2b) and RLmale 1 (H-2d) leukemias were solubilized with deoxycholate and partially purified based on their adherence to a Lens culinaris hemagglutinin column. In double reciprocal experiments we have shown that these preparations specifically inhibit conjugate formation between target cells and alloimmune peritoneal lymphocytes, a preparation rich in cytotoxic T lymphocytes.

Effect of spin-labeled maleimide on 14S and 30S dyneins in solution and on demembranated ciliary axonemes.

The effects of N-1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide(SLM) on the pellet height response and ATPase activity of glycerinated Triton X-100 extracted cilia of Tetrahymena pyriformis have been studied. Preincubation of cilia with SLM caused complete inhibition of the pellet height response and an initial increase in ATPase activity followed upon longer exposure to SLM by inhibition of ATPase. The effect of SLM on extracted 30S dynein was the reverse of that for whole cilia: ATPase activity was increased when 30S dynein was added to a mixture of ATP and SLM and inhibited when the 30S dynein was preincubated with SLM. The activity of 14S dynein was only inhibited by SLM. Electron spin resonance spectra of ciliary axonemes that had reacted with SLM for various times showed that much of the covalently bound SLM was strongly immobilized even after 1 min of reaction, when ATPase activity increased twofold. The proportion of strongly immobilized label increased with longer times of reaction. Addition of ATP to SLM-labeled axonemes caused a small decrease in the height of the spectral peak corresponding to strongly immobilized label as compared with that of weakly immobilized label, indicating an increase in rotational freedom of some covalently bound label. The results suggest that ATP causes a conformation change affecting a sulfhydryl group(s) involved in the mechanochemical system. It was also shown that beta,gamma-methylene ATP(AMP-PCP) is an inhibitor of dynein ATPase. This analogue of ATP is not hydrolyzed by whole cilia or by the extracted dyneins and does not cause a pellet height response. With Mg2+ as divalent cation, AMP-PCP inhibits 30S dynein more than it inhibits 14S dynein; with Ca2+, the inhibition of 30S dynein is reduced, and there is no inhibition of 14S dynein. Under conditions where AMP-PCP inhibited 30S dynein ATPase it was much less effective than ATP in protecting against the loss of ATPase activity by SLM. Although SLM inhibited Mg2+-activated 14S and 30S dyneins in solution, it did not inhibit ciliary ATPase activity. These results support the view that at least 2 SH groups are involved in ciliary motility and that their reactivity to SH reagents depends on whether the dyneins are in situ or have been extracted.

On the use of the spin labeling technique in the study of erythrocyte membranes.

ESR spectra and scanning electron micrographs of human erythrocytes spin labeled with the conventional stearic acid nitroxide substituted at the 5-position have been obtained over a range of label-to-lipid ratios. While morphological changes as previously reported (Bieri, V. G., Wallach, D. F. H. and Lin, P. S. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4797-4801) are reproduced, it is shown that at label-to-lipid ratios of 1:10 or less the basic ESR spectrum is not significantly affected. At low label concentrations the spin labeling technique is a viable one and can be used to investigate membrane properties.